PROJECT SUMMARY/ABSTRACT While the sequence of the genome is invariant across cell types, its function varies widely. As a result, the epigenome, which is composed of histone variants, DNA modification, chromatin structural proteins, and the cadre of proteins that add, remove and interpret post-translational modifications, is of principle importance to understanding the function of cardiac and other cell types. A critical question with basic and translational impact in cardiac physiology is: how does the cell maintain the correct chromatin environment conducive to a healthy adult myocyte transcriptome? In preliminary studies for this renewal, we have adapted methods for chromatin conformation analysis to work in adult cardiac myocytes. We have mapped topologically associating domains (TADs) in myocytes from the basal and pressure overloaded heart, identifying regions of chromatin accessibility and partitioning of genes into active and inactive compartments based on topology. RNA-seq studies have been carried out in these same models to understand the impact on transcription. Mechanistically, we have revealed the actions of chromatin structural proteins in epigenomic specification, which, together with our global measurements of chromatin accessibility and gene expression, serve as the scientific premise for this grant. In addition, we have developed the techniques to determine, with individual locus specificity, how these proteins (including the high mobility group members HMGB2, HMGA1 and HMGN5, plus Nap1 and others identified by proteomics) control local accessibility and transcription through the interaction with other histone marks. We hypothesize that chromatin structure serves as a principal integrator of environmental stress and the other major determinant of all common disease: population scale genetic variability. We propose three aims to test this hypothesis: first, we will determine the fundamental structure-function relationship of the cardiac epigenome in the healthy and diseased heart using Hi-C and RNA-seq. Second, we will determine the logic for transcriptional regulation by chromatin structural proteins at individual loci using a combination of targeted chromatin accessibility analysis, proteomics, ChIP-seq and gain/loss of function. Third, we will identify and validate functional motifs within the epigenome, revealing how genetic variation combines with differential protein binding to specify altered transcription, and thus cardiac phenotype, across a population.